User:Anat Levit/Sandbox 2

Introduction
Human Prokineticin 1 (PK1) and its close homologue PK2 are two secreted proteins, which belong to the AVIT protein family (for example see 1imt, the snake ortologues of human PK1). They are small related peptides of 80-90 amino acids in length, sharing 10 conserved cysteins, which create a five disulphide-bridged motif (colipase fold) and an identical amino-termini – AVIT. PK's are expressed in a wide array of peripheral tissues, including the steroidogenic glands (such as the ovary, testis and adrenal gland), but also in the gastrointestinal tract, nervous system, bladder, bone marrow and prostate. 

PKs exert their biological function through activation of two homologous G-protein coupled receptors (see Wikipedia: G protein-coupled receptors), termed Prokineticin receptor 1 (PROKR1) and Prokineticin receptor 2 (PROKR2). The receptors are made of seven transmembrane α-helices of approximately 30 residues in length (rainbow colored from N to C terminal), which are connected by intra and extracellular loops. The helices are placed in a lipidic environment, while the loop regions are surrounded by aqueous medium. Until recently, our atomic-level understanding of GPCRs has been based on rhodopsin in its inactive state (1f88). In the past couple of years, the field of GPCR structural biology has enjoyed a renaissance, with the publication of three new members: human A2A-Adenosine receptor (3eml), the turkey β1-Adrenergic receptor (2vt4) and the human β2-Adrenergic receptor (2rh1), as well as resolution of the activated state of bovine rhodopsin (3dqb and 3cap) (for review of all avilable structures see Hanson and Stevens 2009).

The amount of structural information available now makes the homology modeling approach, in which the target protein is built starting from the experimentally known 3D structure of a related protein, much more applicable to GPCRs. The structural models of human PROKRs presented here were generated using the I-TASSER server, based on the templates 1l9h, 3eml, 2rh1 for human PROKR1 and 1l9h, 3eml, 1f88, 2rh1 for PROKR2.

The human Prokineticin receptors share 85% sequence homology, which is a high value among known GPCRs. The proteins diverse mainly in their extra and intra-cellular tails.

The prokineticin and their receptors are expressed in various tissues, including the cardiovascular, gastrointestinal, immune, reproductive, endocrine and nervous systems. The receptors have been shown to couple to Gq, Gi and Gs, thereafter mediating intracellular calcium mobilization, phosphorylation of p42/p44 MAPK, AKT and cAMP accumulation, respectively. Receptor activation has been shown to mediate proliferation, anti-apoptosis, differentiation and mobilization of target cells.

The prokineticin receptors have been found to be involved in various pathologies involving the cardiovascular, reproductive, endocrine and nervous systems. Notably, PROKR2 has been found to be mutated in Kallmann syndrome with dilated cardiomyopathy, a hypogonadism caused by a deficiency of gonadotropin-releasing hormone (GnRH) (see Wikipedia: Kallmann syndrome). Except for V331M and R357W (colored cyan ) which are Leu and Asn in PROKR1, respectively, all other residues mutated in PROKR2 are identical in PROKR1 (colored magenta ). Interestingly, two of the mutated residues, W178 (4.50) and P290 (6.50), are two of the most conserved residues in family A GPCRs (restore previous scene ).

Being highly homologues proteins, expressed in the same cell types and having similar nano-molar affinity to common ligands, it is of importance to understand the structural and functional differences between these receptors. The models will help us as we examine the possible differences between the receptors, specifically, differences in post-translational modifications such as receptor phosphorylation, and differences in ligand binding, i.e., binding site identification.

Phosphorylation site variations


Almost all GPCRs are regulated by phosphorylation and this is a key process in determining the signaling properties of these receptors. Receptors are multiply phosphorylated at sites that can occur throughout the intracellular regions of the receptor. It is well established that GPCR phosphorylation is a complex process involving a range of different protein kinases able to phosphorylate the same receptor at different sites and that this results in differential signaling outcomes, which can be tailored in a tissue specific manner to regulate biological processes. Sequence analysis of the PKR subtypes revealed that the majority (63%) of putative phosphor-acceptor residues (Ser, Thr and Tyr) are fully conserved between the subtypes, and the rest are either removed in one of the proteins (23%), i.e., the homologues position does not contain a phosphor-acceptor residue, or is changed to another (11%), for example, a switch from Ser to Thr. We hypothized that differential signaling of the PKR subtypes may result from phosphorylation of homologus residues by different kinases due to presence of phosphovariants in positions surrounding the phosphor-acceptor residue, and not due to change/elimination of this residue between the subtypes.

To this aim, we performed prediction of the putative phosphorylation sites in human PROKR1 and PROKR2 using the GPS, PPSP, NetPhos and NetPhosK webservers. Only sites predicted to be phosphorylated by all methods were considered as putative phosphorylation sites. The phosphovariants can now be identified when the phosphorylation sites or interacting kinases were altered between subtype sequences. The identified sites were classified according to one of the following types, defined by Ryu et al. 2009: Identical phosphor-acceptor residues predicted to be phosphorylated by the same kinases are colored yellow (view all predictions ).
 * 1) In type I the phosphorylation site is in the same location as the variation, and can either create a new phosphorylation site (Type I+) or eliminate an existing site (Type I-) (colored green ).
 * 2) In type II the variation is not in the same location as the phosphorylation site, and can either create a new phosphorylation site (Type II+) or eliminate an existing site (Type II-) (colored blue ).
 * 3) Type III are variations that change only the type of kinase involved, without affecting the phosphorylation site itself (<font color='red'>colored red ).

As seen from the results, homologues residues in the receptors are predicted to be phosphorylated by different kinases, and some of the predicted phospho-sites are receptor unique.

By understanding the flexible nature of PK receptors phosphorylation it may be possible to develop agonists or allosteric modulators that promote a subset of phosphorylation events on the target GPCR and thereby restrict the action of the drug to a particular receptor mediated signaling response.

Binding site analysis
To determine the location of a potential TM binding cavity for each receptor, we structurally aligned and superimposed each of the models onto the structures of bovine Rhodopsin (1l9h, 1f88), human β2-Adrenergic receptor (2rh1) and human A2A-Adenosine receptor (3eml). The Prokineticin receptors binding sites were determined based on homology to the known binding site-composing residues of the templates.

<applet load='PKR1_model1.pdb' size='300' name='PROKR1' frame='true' align='right' caption='Human PROKR1' SCENE='User:Anat_Levit/Sandbox_1/Initial_pkr1/1'/>

The TM cavity of PKR1 and PKR2 is similar to the epinephrine binding site of β2-Adrenergic receptor. It is a narrow and deep cleft that is largely concealed from solvent, which may enable ligand interaction with both walls (via van der Waals contacts). Based on the comparison to 2RH1, we can see that the residues lining the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_2rh1_based_residues/4'>PROKR1 binding site are hydrophobic, which may contribute to potential affinity and polar, which can allow for strong directional constraints through electrostatic interactions. The <scene name='User:Anat_Levit/Sandbox_1/Pkr2_2rh1_residues/5' target='PROKR2'>PROKR2 predicted binding site is almost identical to the PROKR1 site, except for an addition of Ala322 in PROKR2, which is not present in PROKR1, and Glu240 in PROKR1, which is not present in PROKR2.

The ligand binding pocket of bovine Rhodopsin is similar in position to the β2-Adrenergic receptor binding site, and also involves TMs II, III, V and VI. The position does not vary considerably with alternate ligands or between different subtypes of different species. The retinal binding pocket relies mainly on hydrophobic interactions in addition to a covalent linkage with TM VII. In both, <scene name='User:Anat_Levit/Sandbox_1/Pkr1_1l9h_based_residues/3' target='PROKR1'>PROKR1 and <scene name='User:Anat_Levit/Sandbox_1/Pkr2_1l9h_residues/1' target='PROKR2'>PROKR2, Trp288 is part of the binding pocket. This position is homologues to Trp265 (6.48) of Rhodopsin which interacts with retinal (a tryptophan is in general conserved at this position in class A receptors and is thought to be involved in receptor signaling).

The ligand binding pocket of A2A-adenosine receptor assumes a very different location to that of Rhodopsin and β2-Adrenergic receptor. The interface is shifted to TMs VI and VII and there is also extensive interaction with ECL2. However, there is still minor contribution from TMs II, III and V, which is seen in our structural superposition of the

<scene name='User:Anat_Levit/Sandbox_1/Pkr1_3eml_based_residues/1' target='PROKR1'>PROKR1 and <scene name='User:Anat_Levit/Sandbox_1/Pkr2_1l9h_residues/2' target='PROKR2'>PROKR2 models.

Based on this analysis, we defined the core residues which line the <scene name='User:Anat_Levit/Sandbox_1/Pkr1_consensus/2' target='PROKR1'>PROKR1 and <scene name='User:Anat_Levit/Sandbox_1/Pkr2_consensus/2' target='PROKR2'>PROKR2 binding pocket (the selected residues appear in at least two of the superpositions described). The pockets of the two receptors are almost identical, except for the additional Tyr140 and Glu319 residues in PROKR2. Tyr140 is known to be mutated in Kallmann syndrome. The p.Y140X nonsense mutation probably results in a PROKR2 with complete loss of function through the generation of an aberrant transcript that can be unstable or encodes for a truncated protein, lacking the carboxyl terminal domain.

Conclusion
The high conservation of the ligand binding pocket of the prokineticin receptors may explain the very similar affinity of the receptors to their cognate ligands. This has also been observed in other subfamilies of GPCRs (such as dopamine, serotonin, histamine and the adrenergic receptors) and may probably explain the difficulty in obtaining potent subtype-selective compounds in pharmaceutical discovery programs.

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<applet load='PKR2_model1.pdb' size='300' frame='true' name='PROKR2' align='right' caption='Human PROKR2' SCENE='User:Anat_Levit/Sandbox_1/Pkr2_2rh1_residues/2'>